RESUMO
Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that is responsible for significant economic losses worldwide. In spite of its relevance, the limited knowledge about the host immune responses that provide effective protection against the disease has long hampered the development of an effective vaccine. The identification of host proteins with an expression that correlates with protection against bTB would contribute to the understanding of the cattle defence mechanisms against M. bovis infection. In this study, we found that ERAP1 and PDE8A were downregulated in vaccinated cattle that were protected from experimental M. bovis challenge. Remarkably, both genes encode proteins that have been negatively associated with immune protection against bTB.
Assuntos
Bovinos/genética , Bovinos/imunologia , Regulação para Baixo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Mycobacterium bovis/imunologia , Tuberculose Bovina/prevenção & controle , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mycobacterium bovis/patogenicidade , RNA Mensageiro/biossíntese , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/imunologia , VacinaçãoRESUMO
Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg(-1) per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration-responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8-32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , GMP Cíclico/fisiologia , Óxido Nítrico/fisiologia , Pênis/enzimologia , Pênis/fisiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Estimulação Elétrica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Relaxamento Muscular/efeitos dos fármacos , Óxido Nítrico/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , RNA Mensageiro/análise , Transdução de Sinais/efeitos dos fármacos , Citrato de Sildenafila , Sulfonas/farmacologiaRESUMO
Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia , Equilíbrio Hidroeletrolítico , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Expressão Gênica , Microscopia Imunoeletrônica , Estrutura Terciária de Proteína , Vacúolos/químicaRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Trypanosoma cruzi/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Animais , Domínio Catalítico , Células Cultivadas , GMP Cíclico/metabolismo , Amplificação de Genes/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Parasitos/enzimologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência , ortoaminobenzoatos/metabolismoRESUMO
Cyclic nucleotide phosphodiesterases constitute the only known mechanism to inactivate regulatory signals involving cAMP or cGMP. In our laboratory a cAMP-specific phosphodiesterase associated to the flagellar apparatus, named TcPDE1, was identified in Trypanosoma cruzi. By using the catalytic domain sequence of TcPDE1 to screen a Trypanosoma cruzi genomic data base, a novel T. cruzi phosphodiesterase sequence was found and characterized. TcPDE4 encodes a 924-amino acid protein and shows homology with the PDE4 vertebrate subfamily. The sequence shows three conserved domains, FYVE, phosphohydrolase and PDEaseI. The FYVE zinc-finger domain is characteristic of proteins recruited to phosphatidylinosytol 3-phosphate-containing membranes, whereas the two others are characteristic of phosphohydrolases and members of the cyclic nucleotide phosphodiesterases. Sequence analysis shows all characteristic domains present at the type-4 phosphodiesterases specific for cAMP. Moreover, TcPDE4 shows the inhibition profile characteristic for PDE4 subfamily, with an IC50 of 10.46 microM for rolipram and 1.3 microM for etazolate. TcPDE4 is able to complement a heat-shock-sensitive yeast mutant deficient in phosphodiesterase genes. The enzyme is specific for cAMP, Mg(2+)-dependent and its activity is not affected by cGMP or Ca(2+). The association of TcPDE4 with membranes was studied by subcellular fractionation of recombinant yeast and extraction in several conditions. Most of the enzyme remained associated to the membrane fraction after treatment with high salt concentration, detergent, or chaotropic agents. This support previous hypotheses that in this parasite cAMP phosphodiesterases, and consequently cAMP levels, are compartmentalized.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Membrana Celular/enzimologia , Trypanosoma cruzi/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , DNA de Protozoário/análise , Etazolato/farmacologia , Cinética , Dados de Sequência Molecular , Inibidores de Fosfodiesterase/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rolipram/farmacologia , Alinhamento de Sequência , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Trypanosoma cruzi/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/análise , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Clonagem Molecular , Flagelos/enzimologia , Componentes do Gene , Teste de Complementação Genética , Microscopia Confocal , Dados de Sequência Molecular , Frações Subcelulares/química , Leveduras/enzimologia , Leveduras/genéticaRESUMO
We have previously shown that in rat pups intracranially injected with a single dose of apotransferrin (aTf), there is an early oligodendroglial cell OLGc differentiation. The expression of the mRNAs of myelin basic proteins and of 2',3' cyclic nucleotide 3'-phosphodiesterase and the amount of the corresponding proteins, as well as myelin glycolipids and phospholipids, were significantly increased in these animals at 10 and 17 days of age. Microtubules and myelin basic proteins appear to be closely associated in OLGc and it has been shown that the mRNAs of myelin basic proteins are concentrated in the OLGc processes. The aim of this work was to clarify if the accelerated myelination produced by aTf could be linked to changes in certain cytoskeletal elements present in the myelin fraction such as tubulin, actin, and different microtubule-associated proteins (MAPs). A significant increase in the expression of the mRNA of tubulin and actin was observed in the brain of the aTf-treated animals. Several MAPs, particularly MAP 1B and stable tubule only peptide as well as actin and tubulin, were markedly increased in the Triton X-100 insoluble pellet obtained from the myelin fraction of these animals. The changes that we have previously described in the myelin of aTf intracranially injected rats, could be the consequence of its action on the cytoskeletal network of the OLGc. An enlargement of this structure would result in a more efficient and faster movement of the different components that are normally transported to the myelin by the cytoskeleton of this cell.
Assuntos
Apoproteínas/farmacologia , Proteínas do Citoesqueleto/genética , Citoesqueleto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Básica da Mielina/genética , Oligodendroglia/fisiologia , Transferrina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Animais Recém-Nascidos , Apoproteínas/administração & dosagem , Citoesqueleto/metabolismo , Glicolipídeos/metabolismo , Microinjeções , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Fosfolipídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Wistar , Transcrição Gênica/efeitos dos fármacos , Transferrina/administração & dosagemRESUMO
Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP-dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA-dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897-11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total catalytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild-type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1-6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit.